GTPase-activating proteins of ADP-ribosylation factors (ARFGAPs) form a family of proteins that play key roles in cell adhesion and migration, tumor progression, as well as neuronal development. A body of evidence also indicates that ARFGAPs are involved in various diseases, including cancer, Alzheimer's disease, and autism. However, the detailed mechanism by which ARFGAPs regulate different diseases is largely unknown. The long-term goals are to better understand the network of proteins directly modulated by ARFGAPs, and to develop small molecule regulators of distinct ARFGAPs to dissect their biological functions. This particular application is built on the previous discovery of QS11, a small molecule that synergizes with Wnt proteins to activate the Wnt/2-catenin signaling pathway through inhibition of multiple ARFGAPs. The objective is to use and refine QS11 to dissect ARFGAP-regulated cell signaling. Specifically, we will pursue three aims: 1) Define the molecular basis for the inhibition of ARFGAPs by QS11; 2) Identify QS11 analogs with higher potency and selectivity against ARFGAPs; and 3) Define the mechanism by which the inhibition of ARFGAP leads to Wnt synergy. Under the first aim, two complementary approaches will be applied to clarify the mechanistic details of ARFGAP inhibition by QS11. Under the second aim, both library synthesis and rational design will be used to improve QS11's potency and selectivity against ARFGAPs. Under the third aim, biochemical and genetic tools will be used to understand how inhibition of ARFGAPs by QS11 leads to its Wnt synergy. The proposed research is significant, because it will lead to the first sets of small molecule regulators that can temporally and spatially perturb ARFGAP-associated cell signaling, both in cell culture and in whole organisms. Such perturbation is essential to understand the roles for ARFGAPs in diseases. In addition, the proposed research will establish a novel link between ARFGAPs and the Wnt/2-catenin signaling pathway.